This Project This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein. This project explores the potential homologs and orthologs of the NLCR5 protein.
Background Information NLCR5, short for NOD-like receptor family CARD domain containing 5, usually found/ produced in either the nucleus or the cytoplasm. NLRC5, is an intracellular protein that plays an important regulatory role in the immune system. NLRC5 is a pattern recognition receptor implicated in innate immunity to viruses potentially by regulating interferon activity. NLRC5 has also been suggested to play a positive role in the regulation of Major Histocompatibility Class I (MHCI) molecule expression. This aspect of NLRC5 function was further investigated with the help of Nlrc5-deficent by using knockout technique which showed reduced MHCI expression in lymphocytes (particularly with T-cells and Natural Killer cells) (Staehli et al 2012). In lymphocytes, NLRC5 localizes to the nucleus and drives MHCI gene expression by occupying H-2D and H-2K gene promoters.
Figure 1. Interferon (NLCR5) is one class of cytokine released from a variety of cells. A viral-infected cell releases IFN, which stimulates antiviral changes to neighboring cells to prevent their infection and induces macrophages and NK(Natural Killer) cells to destroy viral-infected cells.
Figure 2. Probable spatial structure of the NOD-protein, presented in different ways: A-ball model, B- ribbon model
The NLCR5 gene encodes a member of the caspase recruitment domain-containing NLR family. This gene plays a role in cytokine response and antiviral immunity through its inhibition of NF-kappa-B activation and negative regulation of type I interferon signaling pathways (Neerincx et al 2010). Over production/expression of this particular protein/gene is varily assoiciated with auto-immune disorders while in contrast, under production of this particular gene is associated with a variety of immuno-compromised syndromes (Meyer et. al 2009).
Methods Whale shark predicted orthologs The human protein sequence (ENSP00000262510) was used as query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. Top predicted protein hits were then used as queries (using the full predicted sequence not only the aligned sequence) in protein BLASTs against the NCBI human protein database. The whale shark predicted protein database was also searched using the elephant shark predicted NCLR5 protein sequence as query. Predicted orthologs NLCR5 predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for mouse, zebrafish, African clawed frogs, and elephant shark protein databases. The human NLCR5 protein (ENSP00000262510) was used as query sequence in these searches with default settings. Phylogenetic tree The hit with the lowest E-value for each non-whale shark species search (using the human protein as query) along with the top 2 whale shark BLAST hits were used to create a multiple sequence alignment and phylogenetic tree. ClustalW2 with default settings was used to create the alignment and tree.
Searching for NLRC5 in the whale shark The human NLCR5 protein sequence was used to query the whale shark predicted protein database and results are shown in Table 2. There were 5 hits with E-values below 4e-25 with the next smallest E-value being 6e-31. These 5 best hits were then Blasted against the human protein database using NCBI BLASTp.
ID
Length
Top hit name
E-value
Percent Identities
g46108.t1
510
NOD domain
7e-54
77%
g46616.t1
570
NOD domain
4e-32
27%
g44600.t1
490
NOD domain
6e-31
76%
g31737.t1
339
NOD domain
7e-27
33%
g41147.t1
497
NOD domain
4e-25
26%
Table 1. Human NLCR5 best BLASTp best hits against the whale shark predicted protein database. The Galaxy server was used to query the predicted whale shark protein database using the human NLCR5 protein sequence. The top 5 hits according to E-value are reported here with their database ID and amino acid length. These sequences were also used as queries against the NCBI human protein database. The top hit (according to lowest E-value) name and E-value are reported here.
As none of the predicted proteins from the Blast of the whale shark proteins using the human NLCR5 sequence as query returned NLCR5 as the best hit against the human protein database, we were not confident that any of these five were NLCR5 Orthologs. We then repeated this process using the Elephant Shark predicted NLCR5 protein as query against the whale shark predicted protein database to see if a more closely related species would return different best hits. There were 3 hits with E-values below 1e-32 (the lowest had an E-value of 7e-54) and the next lowest E-value was 4e-32. The top 3 hits did not match up exactly with the specified protein, however there were several isoforms in the same NOD- receptor family that showed up with percent identities greater that 75% and E-values lower than 1e-61 when using the top 4 hits from the search using the human protein as query.
Protein domains Potential NLCR5-like proteins in the whale shark (from BLAST results) all contain a NACHT-domain, and apart of the ABC_ATPase superfamily (Figure 3). This NTPase domain is found in proteins responsible for apoptosis as well as those involved in MHC transcription (Koonin et. al 2000). The NACHT domain proteins contain between 300 and 400 amino acids. They are part of pattern recognition response and play key roles in regulation of innate immune response. NLRs can cooperate with Toll-like receptors and regulate inflammatory and apoptotic response (Doyle et. al 2006). They are found in lymphocytes, macrophages, dendritic cells and also in various epithelial tissue.
Figure 3. Putative domains of whale shark NLCR5 best hit predicted proteins. All of the four best-hit whale shark predicted proteins contain putative NLCR and NACHT-domains as predicted by NCBI BLAST server analyses.
Orthologs The human NLCR5 protein sequence (ENSP00000262510) was used as query in NCBI BLAST searches against individual species' protein databases. We found three orthologues of the NLCR5 proteins in mice, zebra fish, African clawed frogs and elephant sharks.
Species
Name
ID
Length
E-value
Query Coverage
Homo Sapiens
NLCR5
XP_005256258.1
N/A
N/A
100%
African clawed frog*
NLCR-like, class 5
XP_004914291.1
224
2e-63
98%
Mouse
NLCR5
NP_001028379.2
1976
0
100%
Zebra fish
NLCR5
XP 003200494.2
800
2e-113
100%
Elephant shark
NLRC5
XP_007905775.1
808
0
100%
Table 2. Best hits with human NLRC5 protein BLAST. The human NCLR5 sequence was used in protein BLASTs against individual species. Name, ID, length and E-value of the best hit from each search is reported here.
Phylogeny The best hits from protein database searches using the human NLRC5 protein as query were used to create a phylogenetic tree. According to the phylogenetic tree the two whale sharks are share a degree of similarity as they share a recent common ancestor. However, one of the whale shark predicted proteins (g46108.t1) is more closely related to the humans because of their even more recent common ancestor (Figure 4). Despite, finding a NLCR5 ortholog in the elephant shark, it the least related to the human due to further away common ancestor. Figure 4. Phylogenetic tree of NLCR5 best hits. The best hit from BLAST searches of protein databases (or the best 5 hits in the case of whale sharks) were used in the ClustalW2 program to create a phylogenetic tree. Branch lengths represent relative evolutionary time.
Conclusions We were able to identify orthologs in several different species with the NLCR5 protein, specifically mouse, zebrafish, and elephant shark., however we were not able to strongly identify a predicted NLRC5 ortholog in the whale sharks. Despite, finding a NLCR5 ortholog in the elephant shark, the elephant shark was the least related to the human as opposed to the other animals that we used to find orthologs. With the close relation of the whale shark and the elephant shark, it could be suggested that the whale shark does have a portion of the NLRC5 protein, however it has been somehow become evolutionarily diverged from the human and the elephant shark NLRC5 protein. This could be the subject of further research. The lack of whale shark orthologs could also be contributed by the high levels of isoforms of the NOD domain found when comparing the predicted whale shark protein to the human NLCR5 protein. Further research can be done to see what the differences are between these isoforms, and if these difference contribute anything more or less to the health of the whale shark. Research could also be done to explore the presence and annotations of NOD domain proteins in sharks, since given the critical role that NCLR5 plays in the immune cells and the body's ability to fight off invading pathogens, especially viruses, in humans.
References Cummings RD, McEver RP. C-type Lectins. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.
Doyle SL, O'Neill LA (October 2006). "Toll-like receptors: from the discovery of NFkappaB to new insights into transcriptional regulations in innate immunity". Biochem. Pharmacol.72 (9): 1102–1113.
Koischi K, Elsen P et al. (December 2012) Nature Reviews Immunology 12, 813-820
Kuenzel S, Till A, Winkler M et al. (February 2010). "The nucleotide-binding oligomerization domain-like receptor NLRC5 is involved in IFN-dependent antiviral immune responses". J. Immunol.184 (4): 1990–2000.
Koonin EV, Aravind L (May 2000). "The NACHT family - a new group of predicted NTPases implicated in apoptosis and MHC transcription activation".Trends Biochem. Sci.25 (5): 223–4
Natalia V. Kostiuk, Maya B. Belyakova, Dzhulianna V. Leshchenko, Mikhail V. Miniaev, Margarita B. Petrova, Elena A. Kharitonova (December 2012) American Journal of Bioinformatics Research Structural Characterization of the NodD Transcription Factor Staehli, F; Ludigs, K; Heinz, LX; Seguín-Estévez, Q; Ferrero, I; Braun, M; Schroder, K; Rebsamen, M; Tardivel, A; Mattmann, C; MacDonald, HR; Romero, P; Reith, W; Guarda, G; Tschopp, J (April 2012).
This Project
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein. This project explores the potential homologs and orthologs of the NLCR5 protein.
Background Information
NLCR5, short for NOD-like receptor family CARD domain containing 5, usually found/ produced in either the nucleus or the cytoplasm. NLRC5, is an intracellular protein that plays an important regulatory role in the immune system. NLRC5 is a pattern recognition receptor implicated in innate immunity to viruses potentially by regulating interferon activity.
NLRC5 has also been suggested to play a positive role in the regulation of Major Histocompatibility Class I (MHCI) molecule expression. This aspect of NLRC5 function was further investigated with the help of Nlrc5-deficent by using knockout technique which showed reduced MHCI expression in lymphocytes (particularly with T-cells and Natural Killer cells) (Staehli et al 2012). In lymphocytes, NLRC5 localizes to the nucleus and drives MHCI gene expression by occupying H-2D and H-2K gene promoters.
Figure 1. Interferon (NLCR5) is one class of cytokine released from a variety of cells. A viral-infected cell releases IFN, which stimulates antiviral changes to neighboring cells to prevent their infection and induces macrophages and NK(Natural Killer) cells to destroy viral-infected cells.
Figure 2. Probable spatial structure of the NOD-protein, presented in different ways: A-ball model, B- ribbon model
The NLCR5 gene encodes a member of the caspase recruitment domain-containing NLR family. This gene plays a role in cytokine response and antiviral immunity through its inhibition of NF-kappa-B activation and negative regulation of type I interferon signaling pathways (Neerincx et al 2010). Over production/expression of this particular protein/gene is varily assoiciated with auto-immune disorders while in contrast, under production of this particular gene is associated with a variety of immuno-compromised syndromes (Meyer et. al 2009).
Methods
Whale shark predicted orthologs
The human protein sequence (ENSP00000262510) was used as query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. Top predicted protein hits were then used as queries (using the full predicted sequence not only the aligned sequence) in protein BLASTs against the NCBI human protein database. The whale shark predicted protein database was also searched using the elephant shark predicted NCLR5 protein sequence as query.
Predicted orthologs
NLCR5 predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for mouse, zebrafish, African clawed frogs, and elephant shark protein databases. The human NLCR5 protein (ENSP00000262510) was used as query sequence in these searches with default settings.
Phylogenetic tree
The hit with the lowest E-value for each non-whale shark species search (using the human protein as query)
along with the top 2 whale shark BLAST hits were used to create a multiple sequence alignment and phylogenetic tree. ClustalW2 with default settings was used to create the alignment and tree.
Searching for NLRC5 in the whale shark
The human NLCR5 protein sequence was used to query the whale shark predicted protein database and results are shown in Table 2. There were 5 hits with E-values below 4e-25 with the next smallest E-value being 6e-31. These 5 best hits were then Blasted against the human protein database using NCBI BLASTp.
As none of the predicted proteins from the Blast of the whale shark proteins using the human NLCR5 sequence as query returned NLCR5 as the best hit against the human protein database, we were not confident that any of these five were NLCR5 Orthologs. We then repeated this process using the Elephant Shark predicted NLCR5 protein as query against the whale shark predicted protein database to see if a more closely related species would return different best hits. There were 3 hits with E-values below 1e-32 (the lowest had an E-value of 7e-54) and the next lowest E-value was 4e-32. The top 3 hits did not match up exactly with the specified protein, however there were several isoforms in the same NOD- receptor family that showed up with percent identities greater that 75% and E-values lower than 1e-61 when using the top 4 hits from the search using the human protein as query.
Protein domains
Potential NLCR5-like proteins in the whale shark (from BLAST results) all contain a NACHT-domain, and apart of the ABC_ATPase superfamily (Figure 3). This NTPase domain is found in proteins responsible for apoptosis as well as those involved in MHC transcription (Koonin et. al 2000). The NACHT domain proteins contain between 300 and 400 amino acids. They are part of pattern recognition response and play key roles in regulation of innate immune response. NLRs can cooperate with Toll-like receptors and regulate inflammatory and apoptotic response (Doyle et. al 2006). They are found in lymphocytes, macrophages, dendritic cells and also in various epithelial tissue.
Figure 3. Putative domains of whale shark NLCR5 best hit predicted proteins. All of the four best-hit whale shark predicted proteins contain putative NLCR and NACHT-domains as predicted by NCBI BLAST server analyses.
Orthologs
The human NLCR5 protein sequence (ENSP00000262510) was used as query in NCBI BLAST searches against individual species' protein databases. We found three orthologues of the NLCR5 proteins in mice, zebra fish, African clawed frogs and elephant sharks.
Table 2. Best hits with human NLRC5 protein BLAST. The human NCLR5 sequence was used in protein BLASTs against individual species. Name, ID, length and E-value of the best hit from each search is reported here.
Phylogeny
The best hits from protein database searches using the human NLRC5 protein as query were used to create a phylogenetic tree. According to the phylogenetic tree the two whale sharks are share a degree of similarity as they share a recent common ancestor. However, one of the whale shark predicted proteins (g46108.t1) is more closely related to the humans because of their even more recent common ancestor (Figure 4). Despite, finding a NLCR5 ortholog in the elephant shark, it the least related to the human due to further away common ancestor.
Figure 4. Phylogenetic tree of NLCR5 best hits. The best hit from BLAST searches of protein databases (or the best 5 hits in the case of whale sharks) were used in the ClustalW2 program to create a phylogenetic tree. Branch lengths represent relative evolutionary time.
Conclusions
We were able to identify orthologs in several different species with the NLCR5 protein, specifically mouse, zebrafish, and elephant shark., however we were not able to strongly identify a predicted NLRC5 ortholog in the whale sharks. Despite, finding a NLCR5 ortholog in the elephant shark, the elephant shark was the least related to the human as opposed to the other animals that we used to find orthologs. With the close relation of the whale shark and the elephant shark, it could be suggested that the whale shark does have a portion of the NLRC5 protein, however it has been somehow become evolutionarily diverged from the human and the elephant shark NLRC5 protein. This could be the subject of further research. The lack of whale shark orthologs could also be contributed by the high levels of isoforms of the NOD domain found when comparing the predicted whale shark protein to the human NLCR5 protein. Further research can be done to see what the differences are between these isoforms, and if these difference contribute anything more or less to the health of the whale shark. Research could also be done to explore the presence and annotations of NOD domain proteins in sharks, since given the critical role that NCLR5 plays in the immune cells and the body's ability to fight off invading pathogens, especially viruses, in humans.
References
Cummings RD, McEver RP. C-type Lectins. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.
Doyle SL, O'Neill LA (October 2006). "Toll-like receptors: from the discovery of NFkappaB to new insights into transcriptional regulations in innate immunity". Biochem. Pharmacol. 72 (9): 1102–1113.
Neerincx A, Lautz K, Menning M et al. (June 2010). "A Role for the Human Nucleotide-binding Domain, Leucine-rich Repeat-containing Family Member NLRC5 in Antiviral Responses". J Biol Chem 285 (34): 26223–32. doi:10.1074/jbc.M110.109736. PMC 2924034. PMID 20538593.
Koischi K, Elsen P et al. (December 2012) Nature Reviews Immunology 12, 813-820
Kuenzel S, Till A, Winkler M et al. (February 2010). "The nucleotide-binding oligomerization domain-like receptor NLRC5 is involved in IFN-dependent antiviral immune responses". J. Immunol. 184 (4): 1990–2000.
Koonin EV, Aravind L (May 2000). "The NACHT family - a new group of predicted NTPases implicated in apoptosis and MHC transcription activation".Trends Biochem. Sci. 25 (5): 223–4
Natalia V. Kostiuk, Maya B. Belyakova, Dzhulianna V. Leshchenko, Mikhail V. Miniaev, Margarita B. Petrova, Elena A. Kharitonova (December 2012) American Journal of Bioinformatics Research Structural Characterization of the NodD Transcription Factor
Staehli, F; Ludigs, K; Heinz, LX; Seguín-Estévez, Q; Ferrero, I; Braun, M; Schroder, K; Rebsamen, M; Tardivel, A; Mattmann, C; MacDonald, HR; Romero, P; Reith, W; Guarda, G; Tschopp, J (April 2012).